Biomolecules, Volume 13, Issue 8 (August 2023) – 111 articles

The diamondback moth is a detrimental insect pest of brassicaceous crops which was among the first crop insects to be reported as DDT resistant. It has since proven to be significantly resistant to nearly every synthetic insecticide used in the field in many crucifer-producing regions. Due to insecticide control failures in some parts of the world, economically viable crucifer production is now all but impossible. As a result, there has been an increasing effort to identify new compounds with strong pesticidal activity. Cantharidin is one such compound that has been shown to be highly effective against a variety of insect pests. However, its chemical synthesis and potential toxicity to non-target organisms have been a major source of concern. Herein, using rational design approaches, a new series of cantharidin-based verbenone derivatives were synthesized and evaluated for their insecticidal activities against the diamondback moth. Among different compounds screened, compounds 6a, 6h, 6i, and 6q emerged as the most potent compounds exhibiting 100% mortality at a concentration of 100 mg/L after four days. These compounds demonstrated a good anti-feeding effect against the diamondback moth on cabbage leaves. Subsequently, a 3D QSAR study was carried out to identify the key structural features of the synthesized compounds and their correlation with insecticidal activity. Full article

(1) Background: The study aimed to investigate the impact of gold nanoparticles capped with Cornus sanguinea (NPCS) and mixed with a fruit extract (Vaccinum myrtillus L.—VL) on human hepatic stellate cells (LX-2) exposed to TGF-β. (2) Methods: NPCS were characterized by UV-Vis, transmission electron microscopy (TEM), zeta potential measurement, X-ray diffraction (XRD) and energy dispersive spectroscopy (EDX). The cytotoxic effects of VL, NPCS and of the hybrid compounds obtained by mixing the two components in variable proportions (NPCS-VL) were assessed. LDH activity, MDA levels, secretion of inflammation markers, the expression of fibrogenesis markers and collagen I synthesis were estimated after treating the cells with a mixture of 25:25 μg/mL NPCS and VL. (3) Results: TEM analysis showed that NPCS had spherical morphology and homogenous distribution, while their formation and elemental composition were confirmed by XRD and EDX analysis. TGF-β increased cell membrane damage as well as secretion of IL-1β, IL-1α and TLR4. It also amplified the expression of α-SMA and type III collagen and induced collagen I deposition. NPCS administration reduced the inflammation caused by TGF-β and downregulated α-SMA expression. VL diminished LDH activity and the secretion of proinflammatory cytokines. The NPCS-VL mixture maintained IL-1β, IL-1α, TLR4 and LDH at low levels after TGF-β exposure, but it enhanced collagen III expression. (4) Conclusions: The mixture of NPCS and VL improved cell membrane damage and inflammation triggered by TGF-β and mitigated collagen I deposition, but it increased the expression of collagen III, suggestive of a fibrogenetic effect of the hybrid material. Full article

It has been argued that methoxylated stilbenes are better candidates for oral administration than hydroxylated stilbenes, including resveratrol, as they share many biological activities but have better bioavailability. By contrast, they have a disadvantage to consider, i.e., their lower hydrophilic character that leads to precipitation issues in the final product. In this work, we analysed and compared the growth inhibition of colorectal cancer cells of the methoxylated stilbene rhapontigenin and some analogues and overcame potential problems in the development of fortified products by designing inclusion complexes. Among several cyclodextrins, we found the one that best fit the molecule by physicochemical and bioinformatics assays. The stoichiometry and the encapsulation constants with natural and modified cyclodextrins were determined by fluorescence spectroscopy. The most promising complexes were analysed at different temperature and pH conditions, determining the thermodynamic parameters, to discover the optimal conditions for the preparation and storage of the products. The results showed that rhapontigenin solubility and stability were significantly improved, achieving a sevenfold increase in water solubility and maintaining more than 73% of the stilbene after three months. These findings could be of great interest for industries that aim to deliver novel bioactive compounds with higher solubility and lower degradation. Full article

The molecular toxicity of the uranyl ion (UO₂²⁺) in living cells is primarily determined by its high affinity to both native and potential metal-binding sites that commonly occur in the structure of biomolecules. Recent advances in computational and experimental research have shed light on the structural properties and functional impacts of uranyl binding to proteins, organic ligands, nucleic acids, and their complexes. In the present work, we report the results of the computational investigation of the uranyl-mediated loss of DNA-binding activity of PARP-1, a eukaryotic enzyme that participates in DNA repair, cell differentiation, and the induction of inflammation. The latest experimental studies have shown that the uranyl ion directly interacts with its DNA-binding subdomains, zinc fingers Zn1 and Zn2, and alters their tertiary structure. Here, we propose an atomistic mechanism underlying this process and compute the free energy change along the suggested pathway. Our Quantum Mechanics/Molecular Mechanics (QM/MM) simulations of the Zn2-UO₂²⁺ complex indicate that the uranyl ion replaces zinc in its native binding site. However, the resulting state is destroyed due to the spontaneous internal hydrolysis of the U-Cys162 coordination bond. Despite the enthalpy of hydrolysis being +2.8 kcal/mol, the overall reaction free energy change is −0.6 kcal/mol, which is attributed to the loss of domain’s native tertiary structure originally maintained by a zinc ion. The subsequent reorganization of the binding site includes the association of the uranyl ion with the Glu190/Asp191 acidic cluster and significant perturbations in the domain’s tertiary structure driven by a further decrease in the free energy by 6.8 kcal/mol. The disruption of the DNA-binding interface revealed in our study is consistent with previous experimental findings and explains the loss of PARP-like zinc fingers’ affinity for nucleic acids. Full article

In archaea and sulfate-reducing bacteria, heme is synthesized via the siroheme-dependent pathway. The last step of this route is catalyzed by the Radical SAM enzyme AhbD and consists of the conversion of iron-coproporphyrin III into heme. AhbD belongs to the subfamily of Radical SAM enzymes containing a SPASM/Twitch domain carrying either one or two auxiliary iron–sulfur clusters in addition to the characteristic Radical SAM cluster. In previous studies, AhbD was reported to contain one auxiliary [4Fe-4S] cluster. In this study, the amino acid sequence motifs containing conserved cysteine residues in AhbD proteins from different archaea and sulfate-reducing bacteria were reanalyzed. Amino acid sequence alignments and computational structural models of AhbD suggested that a subset of AhbD proteins possesses the full SPASM motif and might contain two auxiliary iron–sulfur clusters (AuxI and AuxII). Therefore, the cluster content of AhbD from Methanosarcina barkeri was studied using enzyme variants lacking individual clusters. The purified enzymes were analyzed using UV/Visible absorption and EPR spectroscopy as well as iron/sulfide determinations showing that AhbD from M. barkeri contains two auxiliary [4Fe-4S] clusters. Heme synthase activity assays suggested that the AuxI cluster might be involved in binding the reaction intermediate and both clusters potentially participate in electron transfer. Full article

Malaria poses a significant global health challenge, resulting in approximately 600,000 deaths each year. Individuals living in regions with endemic malaria have the potential to develop partial immunity, thanks in part to the presence of anti-plasmodium antibodies. As efforts are made to optimize and implement strategies to reduce malaria transmission and ultimately eliminate the disease, it is crucial to understand how these interventions impact naturally acquired protective immunity. To shed light on this, our study focused on assessing antibody responses to a carefully curated library of P. falciparum recombinant proteins (n = 691) using samples collected from individuals residing in a low-malaria-transmission region of Thailand. We conducted the antibody assays using the AlphaScreen system, a high-throughput homogeneous proximity-based bead assay that detects protein interactions. We observed that out of the 691 variable surface and merozoite stage proteins included in the library, antibodies to 268 antigens significantly correlated with the absence of symptomatic malaria in an univariate analysis. Notably, the most prominent antigens identified were P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains. These results align with our previous research conducted in Uganda, suggesting that similar antigens like PfEMP1s might play a pivotal role in determining infection outcomes in diverse populations. To further our understanding, it remains critical to conduct functional characterization of these identified proteins, exploring their potential as correlates of protection or as targets for vaccine development. Full article

Diabetic nephropathy (DN) is one of the most devastating diabetic microvascular complications. It has previously been observed that iron metabolism levels are abnormal in diabetic patients. However, the mechanism by which iron metabolism levels affect DN is poorly understood. This study was designed to evaluate the role of iron-chelator deferoxamine (DFO) in the improvement of DN. Here, we established a DN rat model induced by diets high in carbohydrates and fat and streptozotocin (STZ) injection. Our data demonstrated that DFO treatment for three weeks greatly attenuated renal dysfunction as evidenced by decreased levels of urinary albumin, blood urea nitrogen, and serum creatinine, which were elevated in DN rats. Histopathological observations showed that DFO treatment improved the renal structures of DN rats and preserved podocyte integrity by preventing the decrease of transcripts of nephrin and podocin. In addition, DFO treatment reduced the overexpression of fibronectin 1, collagen I, IL-1β, NF-κB, and MCP-1 in DN rats, as well as inflammatory cell infiltrates and collagenous fibrosis. Taken together, our findings unveiled that iron chelation via DFO injection had a protective impact on DN by alleviating inflammation and fibrosis, and that it could be a potential therapeutic strategy for DN. Full article

Mitochondrial dysfunction plays a pivotal role in numerous complex diseases. Understanding the molecular mechanisms by which the “powerhouse of the cell” turns into the “factory of death” is an exciting yet challenging task that can unveil new therapeutic targets. The mitochondrial matrix protein CyPD is a peptidylprolyl cis-trans isomerase involved in the regulation of the permeability transition pore (mPTP). The mPTP is a multi-conductance channel in the inner mitochondrial membrane whose dysregulated opening can ultimately lead to cell death and whose involvement in pathology has been extensively documented over the past few decades. Moreover, several mPTP-independent CyPD interactions have been identified, indicating that CyPD could be involved in the fine regulation of several biochemical pathways. To further enrich the picture, CyPD undergoes several post-translational modifications that regulate both its activity and interaction with its clients. Here, we will dissect what is currently known about CyPD and critically review the most recent literature about its involvement in neurodegenerative disorders, focusing on Alzheimer’s Disease and Parkinson’s Disease, supporting the notion that CyPD could serve as a promising therapeutic target for the treatment of such conditions. Notably, significant efforts have been made to develop CyPD-specific inhibitors, which hold promise for the treatment of such complex disorders. Full article

The number of patients with nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) is increasing globally and is raising serious concerns regarding the increasing medical and economic burden incurred for their treatment. The progression of NASH to more severe conditions such as cirrhosis and hepatocellular carcinoma requires liver transplantation to avoid death. Therefore, therapeutic intervention is required in the NASH stage, although no therapeutic drugs are currently available for this. Several anti-NASH candidate drugs have been developed that enable treatment via the modulation of distinct signaling cascades and include a series of drugs targeting peroxisome proliferator-activated receptor (PPAR) subtypes (PPARα/δ/γ) that are considered to be attractive because they can regulate both systemic lipid metabolism and inflammation. Multiple PPAR dual/pan agonists have been developed but only a few of them have been evaluated in clinical trials for NAFLD/NASH. Herein, we review the current clinical trial status and future prospects of PPAR-targeted drugs for treating NAFLD/NASH. In addition, we summarize our recent findings on the binding modes and the potencies/efficacies of several candidate PPAR dual/pan agonists to estimate their therapeutic potentials against NASH. Considering that the development of numerous PPAR dual/pan agonists has been abandoned because of their serious side effects, we also propose a repositioning of the already approved, safety-proven PPAR-targeted drugs against NAFLD/NASH. Full article

The possible usefulness of alpha-synuclein (aSyn) determinations in peripheral tissues (blood cells, salivary gland biopsies, olfactory mucosa, digestive tract, skin) and in biological fluids, except for cerebrospinal fluid (serum, plasma, saliva, feces, urine), as a marker of several diseases, has been the subject of numerous publications. This narrative review summarizes data from studies trying to determine the role of total, oligomeric, and phosphorylated aSyn determinations as a marker of various diseases, especially PD and other alpha-synucleinopathies. In summary, the results of studies addressing the determinations of aSyn in its different forms in peripheral tissues (especially in platelets, skin, and digestive tract, but also salivary glands and olfactory mucosa), in combination with other potential biomarkers, could be a useful tool to discriminate PD from controls and from other causes of parkinsonisms, including synucleinopathies. Full article

Red cell diseases encompass a group of inherited or acquired erythrocyte disorders that affect the structure, function, or production of red blood cells (RBCs). These disorders can lead to various clinical manifestations, including anemia, hemolysis, inflammation, and impaired oxygen-carrying capacity. Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms, plays a significant role in the pathophysiology of red cell diseases. In this review, we discuss the most relevant oxidant species involved in RBC damage, the enzymatic and low molecular weight antioxidant systems that protect RBCs against oxidative injury, and finally, the role of oxidative stress in different red cell diseases, including sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, and pyruvate kinase deficiency, highlighting the underlying mechanisms leading to pathological RBC phenotypes. Full article

Solid surfaces have been shown to affect the aggregation and assembly of many biomolecular systems. One important example is the formation of protein fibrils, which can occur on a range of biological and synthetic surfaces. The rate of fibrillation depends on both the protein structure and the surface chemistry, with the different molecular and oligomer structures adopted by proteins on surfaces likely to be crucial. In this paper, the aggregation of the model amyloidogenic peptide, A$\beta$(16–22), corresponding to a hydrophobic segment of the amyloid beta protein on a gold surface is studied using molecular dynamics simulation. Previous simulations of this peptide on gold surfaces have shown that it adopts conformations on surfaces that are quite different from those in bulk solution. These simulations show that this then leads to significant differences in the oligomer structures formed in solution and on gold surfaces. In particular, oligomers formed on the surface are low in beta-strands so are unlike the structures formed in bulk solution. When oligomers formed in solution adsorb onto gold surfaces they can then restructure themselves. This can then help explain the inhibition of A$\beta$(16–22) fibrillation by gold surfaces and nanoparticles seen experimentally. Full article

The pathogenesis of detrusor underactivity (DU) is unclear, and the available therapeutic effects are unsatisfactory. We propose to find key molecules and pathways related to DU based on transcriptome sequencing. A rat model of bilateral pelvic nerve injury (BPNI) was established. Bladder tissues from the sham-operated group, 3 and 28 days after BPNI mapping, were taken for urodynamics, histopathology, and RNA-seq. An enrichment analysis of the screened differential expression genes was performed. Three days after BPNI, the results showed urodynamic features of overflow incontinence, while there was a recovery at 28 days after the operation. Masson staining revealed collagen deposition accompanied by progressive thickening of the smooth muscle layer as DU progressed. RNA-seq results suggested that a total of 1808 differentially expressed genes (DEGs) differed among the groups. RNA-seq and subsequent analysis confirmed that the cell cycle and immune response were significantly activated 3 days after BPNI, while extracellular matrix remodeling occurred 28 days after BPNI. Partial DEGs and pathways were verified by qRT-PCR. Validation of key proteins involved in cell cycle, inflammation, and fibrosis was performed by immunohistochemical staining and western blot, respectively. These molecular expression patterns at different time points after BPNI injury provide valuable insights into the search for therapeutic targets for DU. Full article

Calcium (Ca²⁺) sparks are the elementary events of excitation–contraction coupling, yet they are not explicitly represented in human ventricular myocyte models. A stochastic ventricular cardiomyocyte human model that adapts to intracellular Ca²⁺ ([Ca²⁺]_i) dynamics, spark regulation, and frequency-dependent changes in the form of locally controlled Ca²⁺ release was developed. The 20,000 CRUs in this model are composed of 9 individual LCCs and 49 RyRs that function as couplons. The simulated action potential duration at 1 Hz steady-state pacing is ~0.280 s similar to human ventricular cell recordings. Rate-dependence experiments reveal that APD shortening mechanisms are largely contributed by the L-type calcium channel inactivation, RyR open fraction, and [Ca²⁺]_myo concentrations. The dynamic slow-rapid-slow pacing protocol shows that RyR open probability during high pacing frequency (2.5 Hz) switches to an adapted “nonconducting” form of Ca²⁺-dependent transition state. The predicted force was also observed to be increased in high pacing, but the SR Ca²⁺ fractional release was lower due to the smaller difference between diastolic and systolic [Ca²⁺]_SR. Restitution analysis through the S1S2 protocol and increased LCC Ca²⁺-dependent activation rate show that the duration of LCC opening helps modulate its effects on the APD restitution at different diastolic intervals. Ultimately, a longer duration of calcium sparks was observed in relation to the SR Ca²⁺ loading at high pacing rates. Overall, this study demonstrates the spontaneous Ca²⁺ release events and ion channel responses throughout various stimuli. Full article

Background: Low back pain is a global health problem directly related to intervertebral disc (IVD) degeneration. Senolytic drugs (RG-7112 and o-Vanillin) target and remove senescent cells from IVDs in vitro, improving tissue homeostasis. One drawback of using a single senolytic agent is the failure to target multiple senescent antiapoptotic pathways. This study aimed to determine if combining the two senolytic drugs, o-Vanillin and RG-7112, could more efficiently remove senescent cells and reduce the release of inflammatory factors and pain mediators in cells from degenerating human IVDs than either drug alone. Methods: Preliminary data evaluating multiple concentrations of o-Vanillin and RG-7112 led to the selection of four treatment groups. Monolayer and pellet cultures of cells from painful degenerate IVDs were exposed to TLR-2/6 agonist. They were then treated with the senolytics o-Vanillin and RG7112 alone or combined. p16^ink4a, Ki-67, caspase-3, inflammatory mediators, and neuronal sprouting were assessed. Results: Compared to the single treatments, the combination of o-Vanillin and RG-7112 significantly reduced the amount of senescent IVD cells, proinflammatory cytokines, and neurotrophic factors. Moreover, both single and combination treatments significantly reduced neuronal sprouting in rat adrenal pheochromocytoma (PC-12 cells). Conclusions: Combining o-Vanillin and RG-7112 greatly enhanced the effect of either senolytic alone. Together, these results support the potential of senolytics as a promising treatment for IVD-related low back pain. Full article

Xenon (Xe) has shown great potential as a stroke treatment due to its exceptional ability to protect brain tissue without inducing side effects. We have previously developed Xe-loaded liposomes for the ultrasound-activated delivery of Xe into the cerebral region and demonstrated their therapeutic efficacy. At present, the sole FDA-approved thrombolytic agent for stroke treatment is recombinant tissue plasminogen activator (rtPA). In this study, we aimed to investigate the potential of combining Xe-liposomes with an intravenous rtPA treatment in a clinically relevant embolic rat stroke model. We evaluated the combinational effect using an in vitro clot lysis model and an in vivo embolic middle cerebral artery occlusion (eMCAO) rat model. The treatment groups received intravenous administration of Xe-liposomes (20 mg/kg) at 2 h post-stroke onset, followed by the administration of rtPA (10 mg/kg) at either 2 or 4 h after the onset. Three days after the stroke, behavioral tests were conducted, and brain sections were collected for triphenyltetrazolium chloride (TTC) and TUNEL staining. Infarct size was determined as normalized infarct volume (%). Both in vitro and in vivo clot lysis experiments demonstrated that Xe-liposomes in combination with rtPA resulted in effective clot lysis comparable to the treatment with free rtPA alone. Animals treated with Xe-liposomes in combination with rtPA showed reduced TUNEL-positive cells and demonstrated improved neurological recovery. Importantly, Xe-liposomes in combination with late rtPA treatment reduced rtPA-induced hemorrhage, attributing to the reduction of MMP9 immunoreactivity. This study demonstrates that the combined therapy of Xe-liposomes and rtPA provides enhanced therapeutic efficacy, leading to decreased neuronal cell death and a potential to mitigate hemorrhagic side effects associated with late rtPA treatment. Full article

The molecular profiling of circulating tumor DNA (ctDNA) is a helpful tool not only in cancer treatment, but also in the early detection of relapse. However, the clinical interpretation of a ctDNA negative result remains challenging. The characterization of circulating nucleosomes (carrying cell-free DNA) and associated epigenetic modifications (playing a key role in the tumorigenesis of different cancers) may provide useful information for patient management, by supporting the contributive value of ctDNA molecular profiling. Significantly elevated concentrations of H3K27Me3 nucleosomes were found in plasmas at the diagnosis, and during the follow-up, of NSCLC patients, compared to healthy donors (p-value < 0.0001). By combining the H3K27Me3 level and the ctDNA molecular profile, we found that 25.5% of the patients had H3K27Me3 levels above the cut off, and no somatic alteration was detected at diagnosis. This strongly supports the presence of non-mutated ctDNA in the corresponding plasma. During the patient follow-up, a high H3K27Me3-nucleosome level was found in 15.1% of the sample, despite no somatic mutations being detected, allowing the identification of disease progression from 43.1% to 58.2% over molecular profiling alone. Measuring H3K27Me3-nucleosome levels in combination with ctDNA molecular profiling may improve confidence in the negative molecular result for cfDNA in lung cancer at diagnosis, and may also be a promising biomarker for molecular residual disease (MRD) monitoring, during and/or after treatment. Full article

Many diseases in the human body are related to the level of L-cysteine. Therefore, it is crucial to establish an efficient, simple and sensitive platform for L-cysteine detection. In this work, we synthesized platinum palladium bimetallic nanoparticles (Van-Pt_m/Pd_n NPs) using vancomycin hydrochloride (Van) as a stabilizer, which exhibited high oxidase-like catalytic activity. In addition, the catalytic kinetics of the Van-Pt₁/Pd₁ NPs followed the typical Michaelis–Menten equation, exhibiting a strong affinity for 3,3′,5,5′-tetramethylbenzidine substrates. More importantly, we developed a simple and effective strategy for the sensitive colorimetric detection of L-cysteine using biocompatible Van-Pt₁/Pd₁ NPs. The detection limit was low, at 0.07 μM, which was lower than the values for many previously reported enzyme-like detection systems. The colorimetric method of the L-cysteine assay had good selectivity. The established method for the detection of L-cysteine showed promise for biomedical analysis. Full article

Ovarian clear cell (OCCC) and endometrioid (EnOC) carcinomas are often subsumed under the umbrella term “endometriosis-associated ovarian cancer” (EAOC), since they frequently arise from ectopic endometrium settled in the ovaries. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is known to be aberrantly activated both in endometriosis and EAOC; however, its role in the progression of endometriosis to ovarian cancer remains unclear. In fact, cancer-associated alterations in the mTOR pathway may be found in normal uterine epithelium, likely acting as a first step towards ovarian cancer, through the intermediary stage of endometriosis. This review aims to summarize the current knowledge regarding mTOR signaling dysregulation in the uterine endometrium, endometriosis, and EAOC while focusing on the interconnections between the PI3K/AKT/mTOR pathway and other signaling molecules that give rise to synergistic molecular mechanisms triggering ovarian cancer development in the presence of endometriosis. Full article

Branched-chain amino acids are critical metabolic intermediates that can indicate increased risk of cardiometabolic disease when levels are elevated or, alternatively, suggest sufficient mitochondrial energy metabolism and reserve in old age. The interpretation of BCAA levels can be context-dependent, and it remains unclear whether abnormal levels can inform prognosis. This prospective longitudinal study aimed to determine the interrelationship between mortality hazard and fasting serum BCAA levels among older men and women aged ≥65 years with or without hypertension and diabetes mellitus. At baseline (0Y), fasting serum BCAA concentration in 2997 community-living older men and women were measured. Approximately 14 years later (14Y), 860 study participants returned for repeat measurements. Deaths were analysed and classified into cardiovascular and non-cardiovascular causes using International Classification of Diseases codes. Survival analysis and multivariable Cox regression were performed. During a median follow-up of 17Y, 971 (78.6%) non-cardiovascular and 263 (21.4%) cardiovascular deaths occurred among 1235 (41.2%) deceased (median age, 85.8 years [IQR 81.7–89.7]). From 0Y to 14Y, BCAA levels declined in both sexes, whereas serum creatinine concentration increased (both p < 0.0001). In older adults without hypertension or diabetes mellitus, the relationship between mortality hazard and BCAA level was linear and above-median BCAA levels were associated with improved survival, whereas in the presence of cardiometabolic disease the relationship was U-shaped. Overall, adjusted Cox regression determined that each 10% increment in BCAA concentration was associated with a 7% (p = 0.0002) and 16% (p = 0.0057) reduction in mortality hazard estimated at 0Y and 14Y, respectively. Our findings suggested that abnormally high or low (dyshomeostatic) BCAA levels among older adults with hypertension and/or diabetes mellitus were associated with increased mortality, whereas in those with neither disease, increased BCAA levels was associated with improved survival, particularly in the oldest-old. Full article

Intracranial and extracranial large-artery atherosclerosis (LAA) are a main cause of ischemic stroke. Biomarkers may aid in the diagnosis of LAA and help to stratify patients’ risk of stroke. We performed a narrative review of the literature, mainly published in the last five years, with the aim of identifying biomarkers associated either with intracranial or extracranial LAA in humans. Several potential biomarkers of LAA, mainly related to lipidic pathways and inflammation, have been studied. Diagnostic biomarkers of LAA were evaluated by measuring biomarkers levels in patients with LAA stroke and other stroke etiologies. Some biomarkers were associated with the functional prognosis of LAA stroke patients. Increased levels of IL-6 and sLOX-1 were associated with a risk of progression of carotid atherosclerotic disease. Findings support the notion that the immune system plays a central role in the pathogenesis of LAA. Overall, in most studies, results were not externally validated. In the future, biomarkers could be useful for the selection of patients for clinical trials. To adopt these biomarkers in clinical practice, we will need robust multicentric studies proving their reproducibility and a clear practical applicability for their use. Full article

Mesenchymal stem cells (MSCs) are a type of versatile adult stem cells present in various organs. These cells give rise to extracellular vesicles (EVs) containing a diverse array of biologically active elements, making them a promising approach for therapeutics and diagnostics. This article examines the potential therapeutic applications of MSC-derived EVs in addressing neurodegenerative disorders such as Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD). Furthermore, the present state-of-the-art for MSC-EV-based therapy in AD, HD, PD, ALS, and MS is discussed. Significant progress has been made in understanding the etiology and potential treatments for a range of neurodegenerative diseases (NDs) over the last few decades. The contents of EVs are carried across cells for intercellular contact, which often results in the control of the recipient cell’s homeostasis. Since EVs represent the therapeutically beneficial cargo of parent cells and are devoid of many ethical problems connected with cell-based treatments, they offer a viable cell-free therapy alternative for tissue regeneration and repair. Developing innovative EV-dependent medicines has proven difficult due to the lack of standardized procedures in EV extraction processes as well as their pharmacological characteristics and mechanisms of action. However, recent biotechnology and engineering research has greatly enhanced the content and applicability of MSC-EVs. Full article

Inherited bone marrow failure syndromes (IBMFSs) include Fanconi anemia, Diamond–Blackfan anemia, Shwachman–Diamond syndrome, dyskeratosis congenita, severe congenital neutropenia, and other rare entities such as GATA2 deficiency and SAMD9/9L mutations. The IBMFS monogenic disorders were first recognized by their phenotype. Exome sequencing has validated their classification, with clusters of gene mutations affecting DNA damage response (Fanconi anemia), ribosome structure (Diamond–Blackfan anemia), ribosome assembly (Shwachman–Diamond syndrome), or telomere maintenance/stability (dyskeratosis congenita). The pathogenetic mechanisms of IBMFSs remain to be characterized fully, but an overarching hypothesis states that different stresses elicit TP53-dependent growth arrest and apoptosis of hematopoietic stem, progenitor, and precursor cells. Here, we review the IBMFSs and propose a role for pro-inflammatory cytokines, such as TGF-β, IL-1β, and IFN-α, in mediating the cytopenias. We suggest a pathogenic role for cytokines in the transformation to myeloid neoplasia and hypothesize a role for anti-inflammatory therapies. Full article

The melanocortin-4 receptor (MC4R) is essential for the modulation of energy balance and reproduction in both fish and mammals. Rainbow trout (Oncorhynchus mykiss) has been extensively studied in various fields and provides a unique opportunity to investigate divergent physiological roles of paralogues. Herein we identified four trout mc4r (mc4ra1, mc4ra2, mc4rb1, and mc4rb2) genes. Four trout Mc4rs (omMc4rs) were homologous to those of teleost and mammalian MC4Rs. Multiple sequence alignments, a phylogenetic tree, chromosomal synteny analyses, and pharmacological studies showed that trout mc4r genes may have undergone different evolutionary processes. All four trout Mc4rs bound to two peptide agonists and elevated intracellular cAMP levels dose-dependently. High basal cAMP levels were observed at two omMc4rs, which were decreased by Agouti-related peptide. Only omMc4rb2 was constitutively active in the ERK1/2 signaling pathway. Ipsen 5i, ML00253764, and MCL0020 were biased allosteric modulators of omMc4rb1 with selective activation upon ERK1/2 signaling. ML00253764 behaved as an allosteric agonist in Gs-cAMP signaling of omMc4rb2. This study will lay the foundation for future physiological studies of various mc4r paralogs and reveal the evolution of MC4R in vertebrates. Full article

The eukaryotic actin cytoskeleton comprises the protein itself in its monomeric and filamentous forms, G- and F-actin, as well as multiple interaction partners (actin-binding proteins, ABPs). This gives rise to a temporally and spatially controlled, dynamic network, eliciting a plethora of motility-associated processes. To interfere with the complex inter- and intracellular interactions the actin cytoskeleton confers, small molecular inhibitors have been used, foremost of all to study the relevance of actin filaments and their turnover for various cellular processes. The most prominent inhibitors act by, e.g., sequestering monomers or by interfering with the polymerization of new filaments and the elongation of existing filaments. Among these inhibitors used as tool compounds are the cytochalasans, fungal secondary metabolites known for decades and exploited for their F-actin polymerization inhibitory capabilities. In spite of their application as tool compounds for decades, comprehensive data are lacking that explain (i) how the structural deviances of the more than 400 cytochalasans described to date influence their bioactivity mechanistically and (ii) how the intricate network of ABPs reacts (or adapts) to cytochalasan binding. This review thus aims to summarize the information available concerning the structural features of cytochalasans and their influence on the described activities on cell morphology and actin cytoskeleton organization in eukaryotic cells. Full article

The loss of ocular surface (OS) homeostasis characterizes the onset of dry eye disease. Resilience defines the ability to withstand this threat, reflecting the ability of the ocular surface to cope with and bounce back after challenging events. The coping capacity of the OS defines the ability to successfully manage cellular stress. Cellular stress, which is central to the outcome of the pathophysiology of dry eye disease, is characterized by intensity, continuity, and receptivity, which lead to the loss of homeostasis, resulting in a phase of autocatalytic dysregulation, an event that is not well-defined. To better define this event, here, we present a model providing a potential approach when homeostasis is challenged and the coping capacities have reached their limits, resulting in the stage of heterostasis, in which the dysregulated cellular stress mechanisms take over, leading to dry eye disease. The main feature of the proposed model is the concept that, prior to the initiation of the events leading to cellular stress, there is a period of intense activation of all available coping mechanisms preventing the imminent dysregulation of ocular surface homeostasis. When the remaining coping mechanisms and resilience potential have been maximally exploited and have, finally, been exceeded, there will be a transition to manifest disease with all the well-known signs and symptoms, with a shift to allostasis, reflecting the establishment of another state of balance. The intention of this review was to show that it is possibly the phase of heterostasis preceding the establishment of allostasis that offers a better chance for therapeutic intervention and optimized recovery. Once allostasis has been established, as a new steady-state of balance at a higher level of constant cell stress and inflammation, treatment may be far more difficult, and the potential for reversal is drastically decreased. Homeostasis, once lost, can possibly not be fully recovered. The processes established during heterostasis and allostasis require different approaches and treatments for their control, indicating that the current treatment options for homeostasis need to be adapted to a more-demanding situation. The loss of homeostasis necessarily implies the establishment of a new balance; here, we refer to such a state as allostasis. Full article

Glycosylation of IgG regulates the effector function of this antibody in the immune response. Glycosylated IgG is a potent therapeutic used for both research and clinical purposes. While there is ample research on how different cell culture conditions affect IgG glycosylation, the data are missing on the stability of IgG glycome during long cell passaging, i.e., cell “aging”. To test this, we performed three independent time course experiments in FreeStyle 293-F cells, which secrete IgG with a human-like glycosylation pattern and are frequently used to generate defined IgG glycoforms. During long-term cell culturing, IgG glycome stayed fairly stable except for galactosylation, which appeared extremely variable. Cell transcriptome analysis revealed no correlation in galactosyltransferase B4GALT1 expression with galactosylation change, but with expression of EEF1A1 and SLC38A10, genes previously associated with IgG galactosylation through GWAS. The FreeStyle 293-F cell-based system for IgG production is a good model for studies of mechanisms underlying IgG glycosylation, but results from the present study point to the utmost importance of the need to control IgG galactosylation in both in vitro and in vivo systems. This is especially important for improving the production of precisely glycosylated IgG for therapeutic purposes, since IgG galactosylation affects the inflammatory potential of IgG. Full article

Bacterial keratitis in animals presents challenges due to ocular structural barriers, hindering effective drug delivery. In this study, we used biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) to encapsulate the naturally occurring antimicrobial peptide OH-CATH30, an alternative to conventional antibiotics, for the treatment of bacterial keratitis in animals. Microspheres (MS) were prepared using a modified water-in-oil-in-water (W/O/W) double-emulsion method with optimized osmotic pressure. We conducted comprehensive evaluations, including in vitro characterization, encapsulation efficiency determination, in vitro release kinetics, and in vivo/vitro assessments of irritation and bacterial inhibition. The optimized method yielded microspheres with impressive encapsulation efficiency of 75.2 ± 3.62% and a loading capacity of 18.25 ± 5.73%, exhibiting a well-defined particle size distribution (200–1000 nm) and a ζ-potential of −17.3 ± 1.91 mV. The microspheres demonstrated initial burst release followed by sustained and controlled release in vitro. Both in vitro and in vivo tolerance tests confirmed the biocompatibility of the drug-loaded microspheres, as they did not elicit significant irritation in ocular tissues. Remarkable antibacterial effects were observed in both in vitro and in vivo experiments. Our developed PLGA microspheres show promise as an alternative therapeutic option for topical administration in managing keratitis, offering exceptional drug delivery capabilities, improved bioavailability, and potent antibacterial efficacy. Full article

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products biosynthesized from a genetically encoded precursor peptide. The enzymes that install the post-translational modifications on these peptides have the potential to be useful catalysts in the production of natural-product-like compounds and can install non-proteogenic amino acids in peptides and proteins. However, engineering these enzymes has been somewhat limited, due in part to limited structural information on enzymes in the same families that nonetheless exhibit different substrate selectivities. Despite AlphaFold2’s superior performance in single-chain protein structure prediction, its multimer version lacks accuracy and requires high-end GPUs, which are not typically available to most research groups. Additionally, the default parameters of AlphaFold2 may not be optimal for predicting complex structures like RiPP biosynthetic enzymes, due to their dynamic binding and substrate-modifying mechanisms. This study assessed the efficacy of the structure prediction program ColabFold (a variant of AlphaFold2) in modeling RiPP biosynthetic enzymes in both monomeric and dimeric forms. After extensive benchmarking, it was found that there were no statistically significant differences in the accuracy of the predicted structures, regardless of the various possible prediction parameters that were examined, and that with the default parameters, ColabFold was able to produce accurate models. We then generated additional structural predictions for select RiPP biosynthetic enzymes from multiple protein families and biosynthetic pathways. Our findings can serve as a reference for future enzyme engineering complemented by AlphaFold-related tools. Full article